The afferents to serotonin (5HT) containing cells of the median raphe nucleus are known to include GABA, a substance P and met enkephalin. The effect of injecting these substances into the median raphe nucleus was studied on serotonin turnover. GABA had a tonic inhibitory effect on 5HT activity; substance P and met enkephalin both had an excitatory effect on 5HT activity. Since GABA interneurons might be involved in control of serotonergic activity, we also examined the effect of substance P on the turnonver of GABA in the median raphe nucleus. As an estimation of GABA turnover, we measured the accumulation of GABA after inhibition of its catabolism with gabaculine. Stereotaxic injections of this drug in the median raphe caused an increase in GABA that was linear with time for up to 90 min, and rapid in onset. Substance P injection into the median raphe increased the rate of accumulation of GABA by 30%. The data suggest that the predominant effect of substance P is directly on 5HT cell bodies, and does not act via a GABA interneuron.